Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
  • C07D295/02—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms containing only hydrogen and carbon atoms in addition to the ring hetero elements
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
  • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
  • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
  • C07D295/096—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/02—Nutrients, e.g. vitamins, minerals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
  • C07C309/78—Halides of sulfonic acids
  • C07C309/79—Halides of sulfonic acids having halosulfonyl groups bound to acyclic carbon atoms
  • C07C309/83—Halides of sulfonic acids having halosulfonyl groups bound to acyclic carbon atoms of a carbon skeleton substituted by nitrogen atoms, not being part of nitro or nitroso groups

Definitions

• the present invention relates to novel ethanesulfonamide derivatives in which the sulfonamide moiety is bonded to a 3-(cycloalkylaminomethyl)phenoxyalkylamine.
• the ethanesulfonamide derivatives are useful as antisecretory agents which can be used in the treatment of peptic ulcer disease.
• the present invention is directed to ethanesulfonamide compounds of the formula ##STR1## where
• R 1 may be hydrogen or C 1 -C 3 alkyl
• R 2 may be hydrogen or a pharmaceutically acceptable alkali or alkaline earth metal ion such as sodium, potassium, calcium or magnesium;
• R 3 may be hydrogen, Cl, Br, F, C 1 -C 6 alkyl
• R 5 and R 6 are the same or different and may be hydrogen or C 1 -C 3 alkyl
• R 7 may be hydrogen or C 1 -C 6 alkyl
• X may be O, NR 8 , CHR 8 or --(CH 2 ) n --;
• R 8 may be C 1 -C 3 alkyl
• n may be 0, 1, 2, or 3; and its physiologically acceptable salts.
• the ethanesulfonamide derivatives are useful as antisecretory agents which can be used in the treatment of peptic ulcer disease.
• the invention in its broadest aspects relates to ethanesulfonamide derivatives which have antisecretory activity in mammals.
• the ethanesulfonamide compounds of the invention demonstrating antisecretory activity are shown in the above formula.
• R 1 and R 2 are hydrogen;
• R 3 is hydrogen, Cl, Br, F, nitro, C 1 -C 6 alkyl, C 3 -C 6 branched-chain alkyl, CF 3 or CO 2 R 7 , and
• R 4 is hydrogen, or R 3 and R 4 are each Cl or CF 3;
• R 7 is C 1 -C 6 alkyl;
• X is --(CH 2 ) n --
• n 1 or 2.
• the ethanesulfonamide derivatives of the present invention are prepared as follows:
• the aniline 1 is obtained as a commercial material or where R 1 is lower alkyl, the aniline is prepared by literature procedures such as G.W. Gribble et al., J.Am. Chem.Soc. 96, 7812 (1974).
• the ethenesulfonyl fluoride 2 is obtained as a commercial material or Prepared by the literature procedure of J.J. Krutak et al., J. Org. Chem. 44, 3847 (1979).
• the aniline 1 wherein R 3 and R 4 may represent hydrogen, 4-bromo, 4-fluoro, 3,4-dichloro, 4-butyl, 4-pentoxy, 4-isopropoxy, 4-chloro-3-trifluoromethyl, 4-nitro, 4-acetylamino, 4-ethoxycarbonyl, or 4-methylmercapto or other substituents, is mixed with ethenesulfonyl fluoride in an inert solvent such as toluene, acetic acid, dioxane, or N,N-dimethylformamide at a temperature of about 100°-130° C. for about three to 72 hours to afford the ethanesulfonyl fluoride 3.
• an inert solvent such as toluene, acetic acid, dioxane, or N,N-dimethylformamide
• the crude sulfonyl fluoride 3 is reacted with the phenoxypropylamines 4, which are known compounds described in the literature, wherein X may be O or NR (Merck and Co. European Patent 40696), wherein R 7 is lower alkyl, or X may be CHR 7 or --(CH 2 ) n --, wherein n may be 0, 1, 2 or 3 (Bristol-Meyers Co., French Patent 2505835).
• the reaction may be done without a solvent or in an inert basic solvent such as triethylamine, pyridine, lutidine, quinoline or 1,8-diazabicyclo[5.4.0]undec-7-ene at about 80° C. to about 120° C.
• the sulfonamide 5 is obtained as a viscous liquid which may be mixed with a pharmaceutically acceptable acid such as, for example, ethanedioic acid, (E)- or (Z)-2-butenedioic acid, butanedioic acid, or HCl, or other acids, in an inert solvent such as methanol, ethanol, isopropanol, acetone or ether to afford an acid salt of sulfonamide 5 as a solid.
• a pharmaceutically acceptable acid such as, for example, ethanedioic acid, (E)- or (Z)-2-butenedioic acid, butanedioic acid, or HCl, or other acids
• an inert solvent such as methanol, ethanol, isopropanol, acetone or ether
• the free-base sulfonamide 5 may also be mixed with a base such as, for example, NaOH, KOH, or Ca(OH) 2 , or other bases, in an inert solvent such as methanol, ethanol or isopropanol to afford sulfonamide 5 after solvent removal as a solid, wherein R 2 is a pharmaceutically acceptable alkali or alkaline earth metal ion.
• a base such as, for example, NaOH, KOH, or Ca(OH) 2 , or other bases
• an inert solvent such as methanol, ethanol or isopropanol
• R 2 is a pharmaceutically acceptable alkali or alkaline earth metal ion.
• the free-base sulfonamide 5, wherein R 3 is NHCOCH 3 may be treated with one equivalent of NaOH or KOH in an inert solvent such as methanol or ethanol to Produce the sulfonamide 5, wherein R 3 is NH 2 .
• compositions containing a compound of the present invention as the active ingredient in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques
• the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, parenteral, aerosol or topical.
• any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations (such as, for example, suspensions elixirs and solutions) or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
• the carrier will usually comprise sterile water, though other ingredients, for example, to aid solubility or for preservative purposes, may be included
• injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed
• the pharmaceutical compositions will generally contain per dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful and the like, from about 100 to about 2400 mg/kg, and preferably from about 250 to about 800 mg/kg of the active ingredient.
• the substituted ethanesulfonyl fluoride compounds which are employed as intermediates in the Preparation of the ethanesulfonamide derivatives are also active compounds.
• the ethanesulfonyl fluoride intermediates are capable of increasing bone formation and bone mass and as such are useful in the treatment of osteoporosis and related bone-wasting diseases.
• Osteoporosis is a common disabling bone disease which results in the gradual loss of bone mass per unit volume which makes it weak and porous and leads to increased incidence of spine, hip and forearm fractures ["Osteoporosis: Etiology, Diagnosis, and Management", B. Lawrence Riggs and L. Joseph Melton Eds, 1988].
• current medical treatments are only effective in preventing further bone loss (e.g. estrogen replacement, bisphosponates, vitamin D metabolites and calcium supplements).
• the loss in bone mass causes an increase in fractures, which the above preventative treatments are of limited value.
• An ideal goal of therapy in patients with established bone loss is to provide a treatment program that will increase bone mass, i.e. restore lost bone and thus decrease the incidence of fractures and back/joint pain. Wit the exception of sodium fluoride; however, most available treatments fail to increase bone mass.
• Sodium fluoride has been shown to be one agent that increases bone formation and leads to an increase in bone mass in osteoporosis. It is known that fluoride causes proliferation and increases the activity of bone forming cells, osteoblasts (Farley, et al., Science, 1983, 222, 330). Fluoride, together with adequate calcium supplementation, stimulates formation of mineralized bone. Moreover, the rate of bone fracture has been shown to be significantly decreased by fluoride treatment (Riggs, et al., N. Engl. J. Med., 1982, 306, 446). Unfortunately, sodium fluoride has been associated with adverse reactions which include gastrointestinal side effects (nausea, vomiting, diarrhea and bleeding) and rheumatic complications (synovitis and plantar fascial syndrome).
• the ideal treatment of bone los associated with osteoporosis and related bone-wasting diseases should embody an agent which possesses the "bone mitogen" activity of fluoride but lacks its adverse side-effects.
• R may be H, C 1 -C 5 alkyl, C 3 -C 6 branched-chain
• R 1 may be H, C 1 -C 3 alkyl, COR 5 , CO 2 R 5 ,
• R 2 and R 3 may be the same or different and may be
• R 2 and R 3 may be connected to form a 5-, 6- or 7-membered ring containing a methylenedioxy, ethylenedioxy or propylenedioxy group:
• R 4 may be H, C 1 -C 5 alkyl, COR 8 , CO 2 R 8 or CONHR 8 ;
• R 5 may be H or C 1 -C 5 alkyl
• R 6 and R 7 may be the same or different and may be C 1 -C 5 alkyl
• R 8 may be H, C 1 -C 5 alkyl, phenyl or substituted phenyl wherein the substituent is lower alkyl, lower alkoxy, halo, nitro or CF 3;
• n may be 0-4;
• n 0, 1 or 2;
• R 2 when n is zero then R 2 is not H;
• R 4 when n is greater than zero then R 4 is not H;
• the compounds of formulas I and II are useful as cell growth stimulators and in particular as bone mitogens which can be used to treat patients with osteoporosis.
• the novel ethanesulfonyl fluorides include:
• the compounds of formula I are prepared as follows: ##STR6##
• the ethenesulfonyl fluoride was also obtained from a commercial source.
• the amine or aniline used as the starting material is mixed with ethenesulfonyl fluoride in an inert solvent such as toluene, acetic acid, dioxane, or N,N-dimethylformamide at room temperature or about 100°-130° C. depending upon the solvent employed, for 1 to 24 hours to afford the ethanesulfonyl fluoride I.
• an inert solvent such as toluene, acetic acid, dioxane, or N,N-dimethylformamide
• the fluoride compound is then further reacted with an acid halide such as acetyl chloride, propionyl chloride, trimethylacetyl chloride, benzoyl chloride, 4-chlorobenzoyl chloride, or 4-methoxybenzoyl chloride, among others, in an inert solvent such as methylene chloride, dioxane, or tetrahydrofuran in the presence of a base such as pyridine, triethylamine, sodium bicarbonate, 4-dimethylaminopyridine, or DBU among others at room temperature.
• an acid halide such as acetyl chloride, propionyl chloride, trimethylacetyl chloride, benzoyl chloride, 4-chlorobenzoyl chloride, or 4-methoxybenzoyl chloride, among others
• an inert solvent such as methylene chloride, dioxane, or tetrahydrofuran in the presence of a base such as
• Compounds I may also be reacted with various acid anhydrides such as acetic anhydride, propionic anhydride, trimethylacetic anhydride among others in an inert solvent such as methylene chloride, tetrahydrofuran or dioxane at room temperature or refluxing solvent temperature.
• acid halide or acid anhydride reactions produce I where R 4 is COR 8 .
• a halocarbonate such as methyl chloroformate, ethyl chloroformate, butyl chloroformate, or phenyl chloroformate among others in an inert solvent such as methylene chloride, dioxane, or tetrahydrofuran
• a base such as pyridine, triethylamine, sodium bicarbonate
• isocyanates such as methyl isocyanate, ethyl isocyanate, butyl isocyanate, tert-butyl isocyanate, phenyl isocyanate, 4-chlorophenyl isocyanate, or 4-methoxyphenyl isocyanate among others in an inert solvent such as methylene
• R 1 may represent hydrogen, methyl, ethyl, propyl, acetyl, propionyl, methoxycarbonyl, ethoxycarbonyl, aminocarbonyl, methylaminocarbonyl, propylaminocarbonyl, cyano, methylmercapto, methylsulfinyl, methylsulfonyl, ethylsulfinyl or ethylsulfonyl or other substitutents while R 2 and R 3 are methyl, or ethyl or when R 3 is hydrogen, R 2 is methyl or ethyl; and finally R 2 and R 3 may be joined to form a 5-, 6-, or 7-membered ring such as ##STR9##
• compositions containing an ethenesulfonyl fluoride of the present invention as the active ingredient in intimate admixture with a Pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques.
• the pharmaceutical compositions will generally contain per dosage unit, e.g., tablet, capsule, Powder, injection, teaspoonful and the like, from about 0.1 to about 500 mg/kg of the active ingredient.
• the 2-[(4-fluorophenyl)amino]ethenesulfonyl fluoride was produced following the procedure of Example 1 using 4-fluoroaniline (3.33 g, 30 mmol) and ethenesulfonyl fluoride (3.63 g, 33 mmol).
• Example 2 The title compound was produced following the procedure of Example 1 using the above crude ethenesulfonyl fluoride (6.65 g, 30 mmol) and the amine (8.18 g, 33 mmol) in Example 1. The title compound was isolated as a light yellow-green oil in 21.7% yield (2.93 g) after silica gel (200 g) chromatography and was converted to its monooxalate salt in acetone (softens at 115° C., mp 122°-124° C.).
• the crude 2-[(4-ethoxycarbonylphenyl)amino]ethane sulfonyl fluoride was produced following the procedure of Example 1 using ethyl 4-aminobenzoate (4.96 g, 30 mmol) and ethenesulfonyl fluoride (30 mmol) at 120° C. for 24 hours.
• Example 2 The title compound was produced following the procedure of Example 1 using the above ethenesulfonyl fluoride (6.55 g, 23.8 mmol) and the amine (6.49 g, 26.2 mmol) in Example 1.
• the title compound was purified by silica gel (200 g) chromatography and converted to its monooxalate salt in acetone in 21.3% yield (3.01 g) (softens at 126°-134° C., mp >250° C.)
• the crude 2-[(4-nitrophenyl)amino]ethenesulfonyl fluoride was produced following the procedure of Example 1 using 4-nitroaniline (6.91 g, 50 mmol) and ethenesulfonyl fluoride (6.06 g, 66 mmol) at 110° C. for three days.
• Example 2 The title compound was produced following the procedure of Example 1 using the above ethenesulfonyl fluoride (9.88 g, 39.5 mmol) and the amine (10.77 g, 43.4 mmol) in Example 1. The title compound was isolated as an orange-yellow oil in 20% yield (3.80 g) after silica gel (200 g) chromatography and was converted to its monooxalate salt in acetone, mp 140°-142° C.
• the crude 2-[(4-bromophenyl)amino]ethenesulfonyl fluoride was produced following the procedure of Example 1 using 4-bromoaniline (5.16 g, 30 mmol) and ethenesulfonyl fluoride (33 mmol).
• Example 2 The title compound was produced following the procedure of Example 1 using the above ethenesulfonyl fluoride (8.51 g, 30 mmol) and the amine (8.18 g, 33 mmol) in Example 1
• the title compound was Purified by flash silica gel (200 g) chromatography and the resulting golden-brown oil was converted to its monooxalate salt in acetone in 5.6% yield (1.0 g), mp 119°-121° C.
• the crude 2-(phenylamino)ethenesulfonyl fluoride was Produced following the procedure of Example 1 using aniline (2.79 g, 30 mmol) and ethenesulfonyl fluoride (33 mmol).
• Example 2 The title compound was produced following the procedure of Example 1 using the above ethenesulfonyl fluoride (5.76 g, 28.3 mmol) and the amine (7.73 g, 31.2 mmol) in Example 1. The title compound was isolated in 13% yield (1.7 g) after flash silica gel (200 g) chromatography using CH 2 Cl 2 /MeOH/NH 4 OH (96/3.5/0.5), and the resulting yellow gum was converted to its monooxalate salt in acetone, mp 111.5°-115° C.
• Example 2 The title compound was produced following the procedure of Example 1 using the above ethenesulfonyl fluoride (7.86 g, 30 mmol) and the amine (8.18 g, 33 mmol) in Example 1. The title compound was isolated in 7.2% yield (1.7 g) after flash silica gel (200 g) chromatography using CH 2 Cl 2 /MeOH/NH 4 OH (96/3.5/0.5) and was converted to its monooxalate salt in acetone, mp 110°-122° C.
• the crude 2-[(4-pentoxyphenyl)amino]ethenesulfonyl fluoride was produced following the procedure of Example 1 using 4-Pentoxyaniline (10.4 g, 58.1 mmol) and ethenesulfonyl fluoride (60 mmol).
• Example 2 The title compound was produced following the procedure of Example 1 using the above ethenesulfonyl fluoride (10.71 g, 37 mmol) and the amine (10.0 g, 40.7 mmol) in Example 1.
• the title compound was isolated in 12% yield (3.5 g) after flash silica gel (200 g) chromatography using CH 2 Cl 2 /MeOH/NH 4 OH (97/2.5/0.5) and was converted to its dioxalate salt in methanol, mp 156.5°-160° C. (dec).
• Example 2 The title compound was produced following the procedure of Example 1 using the above ethenesulfonyl fluoride (7.7 g, 25 mmol) and the amine (7.4 g, 30 mmol) in Example 1.
• the title compound was isolated in 15% Yield (2.0 g) as a yellow gum after flash silica gel (200 g) chromatography and was converted to its monooxalate salt in acetone. This tan solid was recrystallized from acetone/ether, mp 115°-120° C.
• the crude 2-[(4-butylphenyl)amino]ethenesulfonyl fluoride was produced following the procedure of Example 1 using 4-butylaniline (4.5 g, 30 mmol) and ethenesulfonyl fluoride (33 mmol).
• Example 2 The title compound was produced following the procedure of Example 1 using the above ethenesulfonyl fluoride (30 mmol) and the amine (8.1 g, 33 mmol) in Example 1. The title compound was isolated in 20% yield (3.0 g) as a Yellow oil after flash silica gel (200 g) chromatography using CH 2 Cl 2 /EtOH/NH 4 OH (96/3.5/0.5) and was converted to its dioxalate salt in MeOH/acetone/ ether, mp 115°-119° C.
• Example 2 The title compound was produced following the procedure of Example 1 using the ethenesulfonyl fluoride (3.4 g, 12.5 mmol) of Example 1 and 3-[3-[(4-methylpiperidin-1-yl)methyl]phenoxy]propylamine (3.4 g, 12.9 mmol) at reflux for 20 hours.
• the title compound was isolated in 66.9% yield (4.3 g) as an orange liquid and was converted to its monooxalate salt in acetone/ether, mp 130°-135° C.
• Parietal cells were isolated from the fundic mucosa of rabbit stomachs by a four-stage collagenase digestion Process. The supernatant fraction from the last two stages of this Process contained the individual parietal cells. This cell suspension was centrifuged and reconstituted in a modified Hank's buffer to contain 2-3 ⁇ 10 6 cells/ml. The cells in this suspension were then evaluated for their ability to accumulate 14 C-aminopyrine ( 14 C-AP), a weak base which has been shown to accumulate in acidic environments such as the parietal cell. The accumulation is stimulated by histamine and is blocked by H 2 antagonists. Accumulation of 14 C-AP is also stimulated by dibutyryl cAMP (dbcAMP).
• 14 C-AP 14 C-aminopyrine
• the accumulation is stimulated by histamine and is blocked by H 2 antagonists. Accumulation of 14 C-AP is also stimulated by dibutyryl cAMP (dbcAMP).
• the cells were incubated with 0.5 ⁇ 10 6 cpm 14 C-AP, with various concentrations of histamine or dbcAMP, 1 ⁇ 10 5 M isobutylmethylxanthine, and test compound added in a 20 ⁇ l volume of buffer or dimethylsulfoxide.
• the flasks were incubated in a shaking water bath at 37° C. Two 0.5 ml aliquots were then taken from each flask and cell pellets were collected by centrifugation. The pellets were solubilized with Protosol (NEN) and radioactivity determined by liquid scintillation spectrometry.
• Protosol NNN
• IC 50 concentration of compound required to inhibit 14 C-AP accumulation in the stimulated parietal cell by 50%
• Test compounds were suspended in a 0.5% aqueous solution of methylcellulose (15 cps) and administered intraduodenally (i.d.) at the time of ligation.
• the rats were housed two per cage and sacrificed with CO 2 four hours after ligation.
• the stomachs were removed and contents emptied into a graduated centrifuge tube.
• the tubes were centrifuged, the volume of gastric juice recorded, and any samples obviously contaminated by feces, food or blood eliminated.
• a 1 ml aliquot of gastric juice was titrated with 0.1 N NaOH to a pH of 7.0-7.4.
• Mouse osteoblastic-line cells (MC3T3-El) were plated in Dulbeccos Modified Eagles Medium (DMEM) with 25 mM Hepes Buffer, L-Glutamine, D-Glucose(4.5 gm/1) supplemented with 10% fetal bovine sera, Penicillin (100 units/ml) and Streptomycin (100 ⁇ l) (Penicillin-Streptomycin liquid) and sodium pyruvate (10 ⁇ M final concentration).
• DMEM Dulbeccos Modified Eagles Medium
• L-Glutamine L-Glutamine
• D-Glucose(4.5 gm/1) supplemented with 10% fetal bovine sera
• Penicillin 100 units/ml
• Streptomycin 100 ⁇ l
• sodium pyruvate 10 ⁇ M final concentration
• 3 H-Thymidine (6.7 Ci/mmole) was diluted in DMEM media without FBS and filter sterilized. An aliquot of this solution was delivered to each culture well to
• Test Preparations Solutions of the test compound were made on the day before incubation. These compounds were solubilized in either ethanol or DMSO at a concentration of 100 mM containing 0.1% FBS. The solutions were filter sterilized and then sterile serial dilutions were performed with DMEM media containing 0.1% FBS.
• Osteoblast line cells were plated into 96 well culture plates, 1600 cells in 100 ⁇ L per well in DMEM with 25 mM Hepes Buffer, L-Glutamine, D-Glucose (4.5 gm/L) supplemented with 10% fetal bovine sera, Penicillin (100 units/mL) and Streptomycin (100 ⁇ g/mL) and sodium Pyruvate (10 ⁇ M final concentration).
• the media was replaced with DMEM containing no FBS and the cells were then incubated for an additional 24 hours.
• test compounds were added and screened at concentrations ranging from 10 -10 to 10 -5 M in the Presence of 0.1% FBS. The cells were then incubated an additional 20 hours.
• mice Female Fisher 344 rats, 12 months of age, were housed in climate controlled light/dark (12-hour cycle) rooms and given food and water ad libitum.
• Test Compounds The test compounds were suspended in sesame oil and administered daily via intraperitoneal injections; vehicle treated rats were administered sesame oil along (2 mL/kg).
• [ 3 H]Proline incorporation into bone protein was carried out as previously described (Reddi and Huggins, 1975). [ 3 H]Proline was injected intraperitoneally at a dose of 0.5 ⁇ Ci/g body weight in 0.9% NaCl. Four hours after the injection, rats were bled by cardiac puncture and then killed by CO 2 asphyxiation and the tibia removed and the radioactivity quantified as in Example 28. The results are shown below in Table 4.
• Tritiated tetracycline which is rapidly concentrated in the growing skeleton, can be used as a relative measure of bone forming surface in vivo.
• [ 3 H]Tetracycline was injected intraperitoneally at a dose of 0.125 ⁇ Ci/g body weight in a 0.9% NaCl solution.
• rats were bled by cardiac puncture and then killed by CO 2 asphyxiation and the tibia removed and cleaned of tissue and marrow.
• the tibia are homogenized and digested in 2 N NaOH for 2 hours.
• the precipitate is washed and then hydrolyzed in 6N Hcl.
• An aliquot of the resulting supernatant is analyzed for tritiutum. The results are shown below in Table 4.

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